: Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CFTR that results in a significantly shorter lifespan as well as associated pathologies. Unfortunately, none of the currently available treatments are able to halt the progression of the disease. The predominant CFTR mutation, deltaF508, results in a mutant protein that is retained in the endoplasmic reticulum (ER) and subsequently degraded by the ubiquitin-proteosome pathway. The mechanisms responsible for ER retention of deltaF508 include associations with cytoplasmic chaperone proteins such as Hsc/Hsp70 and Hdj2. We have examined whether over-expression of NBD1 region of CFTRdelta508 containing the phenylalanine deletion could compete for binding to the chaperone proteins, resulting in trafficking of the mutant full-length protein to the cell membrane. We have demonstrated that adenoviral vectors expressing the deltaF508 nucleotide binding domain 1 (deltaF508 NBD1) as well as the NBD1 plus R domain (deltaF508NBD1-RD) could restore Cl- channel activity restoration in infected human CFTR deltaF508 mutant airway cells. However, due to problems with the efficiency of adenoviral transduction of human airway, we have examined the ability to deliver the NBD1-RD domain with a 12 amino acid protein transduction domain (PTD5). In primary human deltaF508 airway cells treated with a control PTD5-eGFP peptide, no significant cAMP stimulated halide efflux was observed, whereas treatment with the PTD5-deltaF508 NBD1-RD fusion restored cAMP-dependent anion permeability as measured by SPQ analysis and patch-clamp. Furthermore, treatment with the PTD5-deltaF508 NBD1-RD fusion reduced the amount of Hdj2 associated with full-length deltaF508 protein. These results suggest that protein-mediated transduction of a fragment of deltaF508 can compete for binding to Hdj2, resulting in release from the ER and trafficking of the mutant CFTR protein to the cell surface. Thus the goal of this Phase I proposal is to establish a rapid, reproducible and quantitative assay an assay based on voltage-sensitive dyes to detect CFTR activity that could be used in the associated Phase II application to map the minimal region in CFTR required to compete for binding to deltaF508. In addition, a second goal of the proposal is to develop methods for purifying soluble PTD-NBD fusion proteins for analysis in the phase II study. The successful completion of the proposed Phase I and Phase II studies should lead to a phase I clinical trial to test the efficacy of this novel PTD-based therapeutic approach to treat CF.